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In the present study, biosynthesis of silver nanoparticles and its antioxidant, antimicrobial and cytotoxic activities were investigated. Silver nanoparticles were extracellularly synthesized using Aspergillus flavus and the formation of nanoparticles was observed after 72 h of incubation. The results recorded from colour changes, UV–vis spectrum and X-ray diffraction (XRD) support the biosynthesis and characterization of silver nanoparticles. UV–vis spectral analysis showed silver surface plasmon resonance band at 420 nm. X-ray diffraction showed that the particles were crystalline with face-centred cubic structure at 45.05°, 65.45° and 78.65° and the size of the silver nanoparticles was 33.5 nm. The synthesized silver nanoparticles showed potent antimicrobial activity against various pathogens, including bacteria and fungi. Biosynthesized silver nanoparticles exhibited strong antioxidant activity as well as cytotoxicity against HL-60 cells in a dose–response relationship. The powerful bioactivity demonstrated by the synthesized silver nanoparticles leads towards the biomedical use as antioxidant, antibacterial and cytotoxic agents.